Seasonal variation of immune response to heterologous erythrocytes in natural populations of red‐backed (Clethrionomys rutilus) and gray‐sided (C. rufocanus) voles in Western Siberia

Abstract We studied the seasonal variation of adaptive humoral immunity (AHI) in northern red‐backed vole (Clethrionomys rutilus Pallas, 1779, RBV) and gray‐sided vole (C. rufocanus Sundevall, 1846, GSV) in Tomsk region of Western Siberia. Immunoresponsiveness (IR) to sheep red blood cells was assessed by the number of antibody‐producing cells in the spleen. The use of a generalized linear model to analyze the effects of species, sex, year of research, and season of withdrawal of individuals from nature on IR showed a significant effect of species identity, season of animal capture, and the interaction of species with season. The RBV demonstrated higher immune responses during a year, and both species had higher IR in winter. Suppression of IR in spring was greater, started earlier, and lasted longer (March–May) in GSV. In RBV, immunosuppression was restricted to April. The significant negative within year correlations of IR with body mass and masses of reproductive organs in GSV indicated a trade‐off between AHI and growth and reproduction processes. A probable explanation for the difference between species in the seasonal variation of AHI may be related to the difference in tropho‐energetic requirements of each vole species. GSV is a predominantly herbivorous rodent and its thermoregulation seems less efficient than of RBV. The deeper spring immunosuppression in GSV may explain in part its higher mortality during the season of colds.


| INTRODUC TI ON
Seasonal variation of immune functions has often been described in vertebrates (Martin et al., 2008), including humans (Dopico et al., 2015;Paynter et al., 2014). According to the winter immunoenhancement hypothesis (Sinclair & Lochmiller, 2000), immune responses among endothermic vertebrates of temperate zones should increase in winter due to the action of evolutionary-determined endogenous bolstering mechanisms (Nelson, 2004;Nelson & Demas, 1996;Sinclair & Lochmiller, 2000) or from the winter decay of the trade-off between reproduction and energetically costly immune functions (Greenman et al., 2005;Martin et al., 2004Martin et al., , 2006. Another possibility of winter enhancement of immunity involves changes in the abundance and distribution of pathogens and parasites over time (Gavier-Widén & Mörner, 1993;Roth et al., 2018). Since contact-transmitted diseases are more common during fall and winter in temperate regions of the world (Nelson, 2004), the enhancement of immune defenses may represent an effort to resist seasonal infections. Differences in life history strategies may also explain immunity enhancement in winter in some species and its absence in others (Lee, 2006;Martin et al., 2008;Nelson, 2004). Another problem is the limited opportunity to record the pattern of winter immunity enhancement in wild populations. The apparent consequences are that the underlying mechanisms observed in nature may overlap, can manifest partly, or may be completely indistinguishable, making it difficult or impossible to explain causality without special experiments. Indeed, the existing evidence for a winter enhancement of immunity based on observations is extremely controversial (Lohmiller & Moshkin, 1999;Martin et al., 2008). The phylogenetic mechanism that boosts the endogenous immune response may be disguised because winter stressors (low temperatures and food shortage) suppress the immune function.
The cessation of reproduction in winter among seasonal breeders may lead to increased immune function through a reduced tradeoff between reproduction and immunity, but this may not become apparent because the action of winter stressors could have an immunosuppressive effect (Martin et al., 2008). Other reasons are also possible. In desert hamsters (Phodopus roborovskii), for instance, the lack of enhancement of adaptive humoral immunity in winter can be explained by higher winter energy metabolism (both basal and maximum metabolic rates), which is also associated with higher production of glucocorticoids (Vasilieva et al., 2020).
Despite the above limitations and negative notions about the low heuristic value of descriptive studies in seasonal variation of immune activity in nature (Martin et al., 2008), a comparison of phylogenetically close species that inhabit the same environment, but differ in certain eco-physiological characteristics, might be valuable. We, therefore, analyzed two species of forest voles from similar habitats in Western Siberia, a region with a severe, strictly seasonal, and sharply continental climate.
We report the results of our study of seasonal variation of adaptive humoral immunity in wintering generations of northern red- Both species overlap much spatially, occur within the same habitats, and demonstrate interspecific overlap in home ranges. They are active under the snow throughout the winter, even at air temperatures of −30° to −40°С, are generally similar in demography and population dynamic (Kravchenko, 1999), and experience a similar load of parasites and infections (Abramov et al., 2011;Krivopalov, 2011;Galbreath et al., 2013). At the same time, red-backed and gray-sided voles differ in their dietary habits and energetic performances. As a result, they behave differently in the winter. The red-backed vole is much more granivorous, while the gray-backed vole feeds mainly on vegetative parts of plants (Hansson, 1985;Koshkina, 1957;Soininen et al., 2013).
At an ambient temperature of 5°С, the red-backed vole demonstrates a higher metabolic rate (144 ml/g/min × 1000) and heat production (42 kcal/kg/h) vs 114 and 34, respectively, in the gray-sided vole (Bashenina, 1977). Less developed mechanisms of heat production and, accordingly, less developed chemical thermoregulation in gray-sided voles (Safronov, 2009) cause the behavioral adaptation for maintaining temperature homeostasis. Gray-sided voles form wintering groups consisting of close relatives, mostly siblings (Ishibashi et al., 1998). In contrast, 60-70% of individuals of red-backed vole overwinter individually (L. B. Kravchenko, unpublished data). The delayed dispersal of juveniles for successful wintering is associated in the gray-sided vole with 1-1.5 months earlier cessation of maturation of underyearlings compared with the red-backed vole (Kravchenko et al., 2012). Based on the above, we assumed that tropho-energetic differences between the two species can affect the seasonal dynamics of immunocompetence, specifically, the energy-costly system of adaptive humoral immunity (Buttgereit et al., 2000;Ots et al., 2001;Shudo & Iwasa, 2001) and could explain some of the demographic differences, in particular the high cold season mortality of gray-sided voles (Hansen et al., 1999;Kusumoto & Saitoh, 2008;Saitoh et al., 2003). During the 3 years of our study, the relative abundance from fall to spring of the red-backed vole varied from 1.1 to 1.8 times compared with 2.3 to 3.6 times in the gray-sided vole (L. B. Kravchenko, unpublished data). We hypothesized that the earlier cessation of maturation in the gray-sided vole compared with the red-backed vole (Kravchenko et al., 2012) and the high mortality of the gray-sided vole during seasons of colds can be related to characteristics of the seasonal dynamics of adaptive humoral immunity. We also examine whether the voles of each species exhibit a pattern of increased humoral immunoresponsiveness to an antigenic challenge in the harsh winter climate of Western Siberia, whether there are differences between species, and, if so, whether these differences can be associated with the species-specific patterns of physiology and behavior.
Both species of forest voles in Siberia are typical "ephemerals" whose reproductive life is limited to one breeding season. Both species are characterized by two seasonal functional groups of individuals with different ontogenetic trajectories (Olenev, 2002), also named "spring and fall cohorts" (Gliwicz, 1996;Gliwicz et al., 1968;Zejda, 1971). From October to April, we caught voles at days with an air temperature not below −10°C. One to two days before catching, the live traps made of wire cloth (8 × 8 × 12) and not tightly covered with polyethylene film were placed into natural under-snow holes visited by voles, under fallen trees, and branches. We identified the presence of voles by their feces. Distance between traps varied from 5 to 10 m. Folded toilet paper (1 m) was placed in the trap as nesting material. Surplus supply of bait consisted of sunflower seeds and carrots. On the day of capture, we opened the traps at 16:00 and examined them at 18:00 and 20:00. In case of capture, the vole, together with the trap, was immediately placed in a foam F I G U R E 1 The studied species: (a) -red-backed, (b) -gray-sided vole. (c) -seasonal temperature variation. Measurements were conducted in the open air, in ground litter, and in soil at 15 cm depth using autonomous temperature recorders (DS1921G-F5, Maxim Integrated Products, USA) every 3 h. The data are averages for every 10 days across 2 years of the registrations.
box (an ordinary thermal box) with plastic bags inside filled with warm (40-45°C) water (10 × 15 × 2 cm). Boxes with traps were transported to animal quarters in the lab. From May to September, we caught voles by live traps in lines with a distance of 5 m between traps. The traps were checked twice a day at 5-6 a.m. and at 7-8 p.m.
Animals we removed from nature were kept individually in cages (25 × 40 × 12 cm) under the natural photoperiod at a temperature of +8-+10°С from October to April, and at natural ambient temperatures for the remainder of the study. Food (oats, apples, grass), water, and nesting material were provided ad libitum.
We weighed the animals immediately after capture within an accuracy of 0.1 g and estimated the masses of the reproductive organs, the testes in males, and the uterus with ovaries in females, with an accuracy of 0.001 g after killing no later than 1 week after capture.
To assess the between-month variation of the masses of reproductive organs in order to increase the sample size, we used data for a longer period: for red-backed vole from 2016 to 2018 (82 males and 59 females), for gray-sided vole from 2016 to 2021 (93 males and 80 females).

| Measurement of adaptive humoral immunoresponsiveness
We immunized voles from 8 to 9 am 36 h after capture. Tests of the duration of the glucocorticoid response to manipulations similar to trapping disturbance and to injection of ACTH in a congeneric species, Clethrionomys glareolus, showed that the effect of the stress factors ends within a day (Rogovin & Naidenko, 2010;Zavjalov et al., 2003). We did not treat pregnant females. To assess adaptive humoral immunity, we used local hemolysis in a liquid medium (Cunningham, 1965). We estimated the number of antibody-producing cells (APC) of the spleen that were formed in response to the introduction of a non-replicating antigen, 0.5 ml of 2% of SRBC suspension injected intraperitoneally. On the 5th day, the voles were killed by cervical dislocation. The suspension of spleen cells was prepared as described by Moshkin et al. (1998).
The reaction mixture consisted of 500 μl spleen cell suspension, 500 μl washed SRBC (4 × 10 9 , erythrocytes/ml), and 500 μl lyophilized guinea pig serum (Biomed, Perm, Russia) resolved with 1 ml of isotonic sodium chloride solution. Cunningham chambers were prepared from glass microscope slides, loaded with 200 μl of reaction mixture (two chambers per individual), and incubated for 2 h at 37°C before hemolysis zones were counted. We calculated and then averaged the number of hemolysis zones (antibodies-producing splenocytes) for each individual. To eliminate within and significant between species differences in body masses, the total number of APCs in the spleen was divided by individual body mass (Novikov et al., 2010). Estimation of the number of APCs per unit of body mass has an advantage over the estimate per unit of spleen mass, since splenomegaly is often observed in forest voles (10.6% in red-backed vole and 7.7% in gray-sided vole in our study). An increase in the mass of the spleen in such cases is not associated with an increase in the number of APCs (Kravchenko, unpublished). The number of APCs per unit of body mass was used as the main indicator of immune activity. To analyze correlations of immunoresponsiveness with body mass and with masses of reproductive organs, we used the absolute number of APCs. groups (normal z-values were computed for each comparison, as well as post hoc probabilities corrected for the number of comparisons for a two-sided test of significance; Siegel & Castellan, 1988). We also used Mann-Whitney U test for independent pair comparisons.

| Statistical procedures
Spearman's Rank-Order Correlation Coefficient (Rs) was used to measure linkage of continuous non-normally distributed data. Since our analyses were based in part on data from a different number of years (we analyzed the between-month variation in the mass of the reproductive organs of voles over a longer time interval, than immunoresponsiveness), we indicated the sample sizes in the headings to the table and figures. Tests were two-sided, with a significance level < 0.05.

| Compliance with regulations when working with animals
We conducted all procedures involving the experimental animals in

| Factors affecting immunoresponsiveness to SRBC
The use of GLZ to analyze the effects of species, sex, year of research, and season of withdrawal of individuals from nature on spleen immunoresponsiveness to antigenic challenge showed significant effects of species identity and season of animal capture.
The only one interaction of predictors with the probability close to the significant level was the interaction of species with season (Table 1).

| Seasonal variation in immune responsiveness to antigenic challenge
The red-backed vole demonstrated higher immunoresponsiveness to SRBS compared with the gray-sided vole during a year (Mann-Whitney U Test: Z = 6.24, N red-backed = 94, N grey-sided = 65, The highest difference between species in immunoresponsiveness was also pronounced in the spring (Mann-Whitney U Test: Z = 4.99, N C.rutilus = 43, N C.rufocanus = 25, p < .001). Regarding differences between months, we found that gray-sided voles showed an earlier onset of spring immunosuppression that started in March and continued to May reaching its lowest median values in May ( Figure 4b). In the red-backed vole, there was no difference between winter months and March, and the lowest median value was in April (Figure 4a). TA B L E 1 Effects of species, sex, year, season of withdrawal (winter, spring, summer end fall) and interaction of species with season on the adaptive humoral immunity response to SRBC in redbacked vole and grey-sided vole. B and SE correspond to parameter estimates and standard errors in GLZ with normal distribution and identity link function; W corresponds to Wald statistic estimates and X 2 corresponds to likelihood ratio test type III. Significant effects (p < .05) are marked in bold. N = 159.

Level of effect
Response and statistics were negative in all cases. In the gray-sided vole, Spearman's Rs were negative and higher, and statistically significant in females (Table 2).

| DISCUSS ION
Season affected the immunoresponse of forest voles caught in nature. We found a statistically significant effect on the magnitude of the immune response to SRBC in both vole species. Both species exhibited a greater immunoresponse in winter with the rather low variance in values. Species differed, however, in their immunoresponses at other times of the year. In the spring, the gray-sided voles showed a suppressed immunoresponse that was deeper, began earlier, and lasted longer than in the red-backed vole. Immunosuppression in the red-backed vole was high but restricted to April.
The year of capture and sex of the voles seemed to have no effect on their immune response. Because the red-backed and graysided voles have promiscuous mating systems (Gromov, 2008), we hypothesized that there may be sex differences in their humoral immune responsiveness to an antigenic challenge (Zuk & Stoehr, 2002), but we were unable to confirm this.
Our results did partly agree with data from other studies that assessed the seasonal immunoresponsiveness to SRBC of bank (C. glareolus) and red-backed voles (Moshkin et al., 1998). The magnitude of the humoral immune response to the antigenic challenge increased in these species of voles from summer to fall. In our study, the immune response in the red-backed vole was higher throughout the year, and the difference in immunoresponsiveness between winter and other seasons was statistically significant; in the gray-sided vole, the difference between winter and other seasons was also significant, but with the exception of fall.
The winter immunity enhancement hypothesis (Nelson, 2004;Nelson & Demas, 1996;Sinclair & Lochmiller, 2000) and the limitations to testing it have been critically discussed in the past (Martin  et al., 2008). As an alternative to the evolutionarily determined endogenous mechanism of enhancing immune activity to counteract winter stressors, the trade-off hypothesis between costs for reproduction and immune defense has been proposed (Greenman et al., 2005;Martin et al., 2004Martin et al., , 2006. These two explanations are not mutually exclusive, and it is impossible to give preference to one or another mechanism without special experiments (Martin et al., 2008). In our case study of two closely related species of forest voles, based on observations, we can only offer hypothetical explanations that seem to us the most plausible.
Traditionally, stress from low temperatures and limited availability of resources is considered to be a factor negatively affecting immunoresponsiveness in winter (Martin et al., 2008;Nelson, 2004).
An increase in the level of corticosterone in September and October in both species of voles was previously revealed (Kravchenko et al., 2016). This could have a negative effect on the immune system during the preparation of physiological systems for winter. However, according to the results of the present study, this does not cause a seasonal decrease in humoral immunoresponsiveness in voles. This may be due to the relatively mild winters of the last two decades.
In Western Siberia, climate warming manifested mainly in winter (Gordov et al., 2011). Winter's increasing mildness in areas with traditionally cold winters can reduce the risk of death and preserve the health of people and livestock (Lacetera, 2019). In rodents, milder winters may be the reason for the lack of winter immunosuppression which could be caused by the low temperature stress (Kusumoto & Saitoh, 2008;Xu & Hu, 2017). In the framework of the winter immunity enhancement hypothesis, it seems possible that the lower The activity of this immune system, according to some estimates, seems to be costly for the organism (Buttgereit et al., 2000;Ots et al., 2001;Shudo & Iwasa, 2001, but see Martin et al., 2008).
In contrast to the red-backed vole, the suppression of immunoresponsiveness in the gray-sided vole in spring was not only greater but it also started earlier (March) and lasted longer (May). These  (Bashenina, 1977) in the predominantly herbivorous gray-sided vole (Koshkina, 1957;Soininen et al., 2013) limits the scope for an intense humoral immune response to antigenic challenges. At the end of April, the wintering groups of relatives (Ishibashi et al., 1998) in the gray-sided vole become disintegrated, and voles form individual home ranges. It has been shown that at 5°C with food provided ad libitum, individual caging of gray-sided voles causes immunosuppression (Kusumoto & Saitoh, 2008). Although we observed a well pronounced between individual variation in immune responsiveness in April, the variation decreased again and the values became minimal in May. The possible reason for the pattern we observed in graysided vole could be a combination of the effects of changing social structure and intensive reproductive effort against the background of still rather low ambient temperatures, not optimal for the species (Figure 1). It is possible that deep and prolonged spring immunosuppression in the gray-sided vole may be one of the reasons for the high mortality during the cold season described for this species (Hansen et al., 1999;Kusumoto & Saitoh, 2008;Saitoh et al., 2003).
Thus, under the conditions of Western Siberia, the differences between two species of forest voles in the depth and duration of spring suppression of adaptive humoral immunity may result from TA B L E 2 Spearman rank correlations of immunoresponsiveness to SRBC (total number of antibody-producing cells, N APC) with body mass and masses of reproductive organs in red-backed and grey-sided voles within a year (2016-2018). Significant effects (p < .05) are marked in bold. N=159.
the specifics of the mechanisms for maintaining temperature homeostasis. The lower level of metabolism and the less developed mechanism of chemical thermoregulation in the mostly herbivorous gray-sided vole (Safronov, 2009)

ACK N OWLED G M ENTS
We thank S.I. Gashkov for help in estimating the temperatures at the habitat of voles. We are very grateful to Prof. Jannet A.
Randal for valuable comments and editing of the English, and we are deeply indebted to both anonymous reviewers and the associate editor for valuable comments and suggestions for improving the manuscript.

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interests. We are following the recent recommendation to use the former genus name of the forest voles, Clethrionomys instead of Myodes (Kryštufek et al., 2020), and use it traditionally for both species, although according to some preliminary data, C. rufocanus can be classified phylogenetically as a separate genus Craseomys (Lebedev et al., 2007)